1. Developmental Biology
  2. Neuroscience

Plexin-B2 controls the timing of differentiation and the motility of cerebellar granule neurons

  1. Eljo Van Battum
  2. Celine Heitz-Marchaland
  3. Yvrick Zagar
  4. Stéphane Fouquet
  5. Rohini Kuner
  6. Alain Chédotal  Is a corresponding author
  1. Sorbonne Université, INSERM, CNRS, Institut de la Vision, France
  2. Pharmacology Institute, Heidelberg University, Germany
https://doi.org/10.7554/eLife.60554
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Cite this article
  1. Eljo Van Battum
  2. Celine Heitz-Marchaland
  3. Yvrick Zagar
  4. Stéphane Fouquet
  5. Rohini Kuner
  6. Alain Chédotal
(2021)
Plexin-B2 controls the timing of differentiation and the motility of cerebellar granule neurons
eLife 10:e60554.
https://doi.org/10.7554/eLife.60554
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10 figures, 8 videos, 1 table and 1 additional file

Figures

Figure 1
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Plexin-B2 expression and generation of cerebellum-specific Plxnb2 conditional knockout models.

(A, B, C), Plexin-B2 protein distribution in the cerebellar cortex during different stages of postnatal development. (A) Plexin-B2 immunostaining on cryostat sections immunolabeled with the Purkinje …

Figure 2
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Developmental time course of cerebellar Plxnb2 phenotype.

The time course of cerebellar foliation and lamination during early postnatal cerebellar development is delayed in Plxnb2 conditional knockout. (A) Pax6 immunostaining labels both pre- and …

Figure 3 with 1 supplement
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Ruffled IGL and ectopic CGN patches in cerebellum-specific Plxnb2 mutant.

(A– D) Whole-mount TO-PRO-3 staining of P14 and P30 cerebella from Plxnb2fl/fl and En1Cre;Plxnb2fl/fl littermates cleared with iDISCO+. In 3D, TO-PRO-3 staining mainly reveals the structure of the …

Figure 3—figure supplement 1
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Difference in cerebellar volume but not motor function.

(A) Quantification of cerebellar volume was done by 3D segmentation of the cerebellum in Imaris. Data is normalized to control cerebellum (dotted line at 100%) P4: 85.64 ± 4.425%. Mann Whitney U …

Figure 3—figure supplement 1—source data 1

Cerebellar volume and function.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
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Proliferating CGNs intermingle with migrating CGNs and have a longer cell-cycle.

(A) Coronal sections of P9 cerebella of Plxnb2fl/fl control and En1Cre;Plxnb2fl/fl littermates injected with EdU 2 hr before perfusion. EdU labels proliferating CGN precursors and Calbindin (CaBP) …

Figure 4—source data 1

Layer thickness and proliferation markers in EGL.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
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No difference in amounts of EdU+ and H3P+ CGNs in EGL.

(A) Graph shows the quantification of the amount of EdU+ cells 2 hr and 24 hr after EdU injection as described in Figure 4D, and the amount of H3P+ cells per μm3 of EGL. EdU+ and H3P+ cells were …

Figure 4—figure supplement 1—source data 1

No difference in amounts of EdU+ and H3P+ CGNs in EGL.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig4-figsupp1-data1-v2.xlsx
Figure 5 with 2 supplements
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Plxnb2 mutant CGNs display aberrant proliferative and tangential stages.

(A) Schematic representation of the in vivo cerebellum electroporation protocol. See Materials and methods for details. (B) Cerebellar sections of electroporated brains at 1 day, 2 days, 1 week and …

Figure 5—source data 1

CGN morphology in vivo and colocalization with proliferation markers.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
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Identity of electroporated cells and quantification of morphological features.

(A) Immunohistochemistry of coronal sections of cerebellum 2 days post-electroporation. GFP shows the electroporated cells and Pax6 marks pre- and post-mitotic CGNs. Graph shows that 99.6 ± 0.21% of …

Figure 5—figure supplement 1—source data 1

Identity of electroporated cells in vivo, morphology of electroporated CGNs.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 2
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Misplaced and misprojecting CGNs keep their identity.

(A) Immunohistochemistry of a sagittal section from an En1Cre;Plxnb2fl/fl cerebellum, more than 3 weeks after electroporation with GFP. GFP labels electroporated CGNs, Calbindin (CaBP) stains …

Figure 6 with 1 supplement
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Abnormal parallel fiber layering in Plxnb2 mutants.

(A) Coronal sections of the cerebellum of P25 mice electroporated with GFP at P7 and re-electroporated with tdTomato (tdTom) at P11. Double immunostaining for GFP and tdTomato. In control (left) the …

Figure 6—source data 1

Parallel fiber distribution.

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Figure 6—figure supplement 1
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Abnormal localization of parallel fiber synapses in Plxnb2 mutant.

(A) Mice were electroporated with GFP at P7 and their cerebellum collected at P9. Sagittal sections were incubated with antibodies against GFP (to label CGNs and parallel fibers), Calbindin (CaBP, …

Figure 6—figure supplement 1—source data 1

Synaptogenesis between parallel fibers and Purkinje cells.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig6-figsupp1-data1-v2.xlsx
Figure 7 with 2 supplements
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Plxnb2 CGNs recapitulate in vitro the developmental defects found in vivo.

(A) EGL explants from P4-P5 wildtype cerebella, fixed after 1, 2, 3, and 5 days in vitro (DIV). Immunocytochemistry for βIII-tubulin and DAPI shows that cells migrate away from the explant and …

Figure 7—source data 1

EGL explants: in vitro CGN morphology.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
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Migrating cells have a CGN identity.

(A) Immunocytochemistry of DIV2 EGL explants for Pax6 (pre- and postmitotic CGNs) with DAPI counterstaining. Cells were counted in 400 × 400 pixel squares close to the explant. Almost all migrating …

Figure 7—figure supplement 1—source data 1

Identity of electroporated cells migrating out of EGL explants.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig7-figsupp1-data1-v2.xlsx
Figure 7—figure supplement 2
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Electroporated cells migrating away from EGL explants have CGN identity.

(A) EGL explants from P4-P5 cerebella electroporated ex vivo with GFP just before culture. Immunocytochemistry for GFP and Pax6 with DAPI staining shows that in both controls and Plxnb2 mutants …

Figure 7—figure supplement 2—source data 1

Identity of cells migrating out of EGL explants.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig7-figsupp2-data1-v2.xlsx
Figure 8
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Plxnb2 mutant CGNs in culture display aberrant tangential migration.

(A) 15-minute-time-lapse confocal still images at t 0, 1, 2, 3, 4, and 5 hr showing GFP+ CGNs migrating from a DIV1 explant (located on the left side of the images). Scale bars 50 μm. (B) Bipolar …

Figure 8—source data 1

EGL explants: live imaging of bipolar CGN migration.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig8-data1-v2.xlsx
Figure 9
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Aberrant proliferation of CGN precursors in Plxnb2 mutant explants.

(A) EGL explants from P4-P5 cerebella at DIV1. Two hr prior to fixation, 10 μM EdU was added to the culture medium. Cultures were stained for EdU, H3P, and DAPI. (B) The number of DAPI+ …

Figure 9—source data 1

EGL explants: in vitro proliferation.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig9-data1-v2.xlsx
Figure 10 with 1 supplement
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Aberrant CGN (precursor) motility before and after division in Plxnb2 mutant explants.

(A, B) Time-lapse confocal imaging series (21 hr) of GFP+ multipolar CGNs in control (A) and Plxnb2 mutant explants at DIV1. (A) In a control, a multipolar cell (outlined in pink at t 0 hr) divides …

Figure 10—source data 1

EGL explants: live imaging of multipolar CGNs.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig10-data1-v2.xlsx
Figure 10—figure supplement 1
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Quantification of the distribution of multi- and bipolar CGNs during time-lapse.

(A) Still images at the beginning (t = 0 hr, DIV1) and at the end (t = 18 hr, DIV2) of a time-lapse imaging series of migrating GFP+ CGNs in EGL explants. Plxnb2 mutant explants show relatively more …

Figure 10—figure supplement 1—source data 1

Morphology of CGNs during live imaging.

https://cdn.elifesciences.org/articles/60554/elife-60554-fig10-figsupp1-data1-v2.xlsx

Videos

Video 1
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3D movie of P4 iDISCO+ cleared Plxnb2fl/fl and En1Cre;Plxnb2fl/fl cerebella.

All cell nuclei are stained with TO-PRO-3, Pax6 and FoxP2-staining is used to visualize CGNs and Purkinje cell bodies, respectively.

Video 2
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3D movie of P14 iDISCO+ cleared Plxnb2fl/fl and En1Cre;Plxnb2fl/fl cerebella.

All cell nuclei are stained with TO-PRO-3.

Video 3
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3D movie of P30 iDISCO+ cleared Plxnb2fl/fl and En1Cre;Plxnb2fl/fl cerebella.

All cell nuclei are stained with TO-PRO-3.

Video 4
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3D movie of P20 iDISCO+ cleared En1Cre;Plxnb2fl/fl cerebellum stained with TO-PRO-3 for all cell nuclei and FoxP2 to visualize Purkinje cell bodies.
Video 5
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3D movie of P65 iDISCO+ cleared Plxnb2fl/fl cerebellum electroporated at P7 with GFP.

Whole mount immunostaining was performed with GFP to stain electroporated CGNs, FoxP2 to visualize Purkinje cell bodies, and TO-PRO-3 to stain all cell nuclei and visualize cerebellar anatomy.

Video 6
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3D movie of P65 iDISCO+ cleared En1Cre;Plxnb2fl/fl cerebellum electroporated at P7 with GFP.

Whole mount immunostaining was performed with GFP to stain electroporated CGNs, FoxP2 to visualize Purkinje cell bodies, and TO-PRO-3 to stain all cell nuclei and visualize cerebellar anatomy.

Video 7
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Representative examples of confocal time-lapse recording of EGL explant cultures of P4-P5 Plxnb2fl/fl and En1Cre;Plxnb2fl/fl cerebella with 15 min interval, starting from DIV1.

Cerebella were electroporated ex vivo with GFP to visualize individual CGNs and follow their migration over time (some striking examples are pseudo-colored). Control CGNs with a bipolar morphology …

Video 8
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Representative examples of confocal time-lapse recording of EGL explant cultures of P4-P5 Plxnb2fl/fl and En1Cre;Plxnb2fl/fl cerebella with 15-min interval, starting from DIV1.

Cerebella were electroporated ex vivo with GFP to visualize individual CGNs and follow their migration over time.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain; strain background (Mus musculus)En1Cre (C57BL/6J)DOI:10.1101/gad.14.11.1377En1tm2(cre)Wrst/J
RRID:IMSR_JAX:007916
Strain; strain background (Mus musculus)Wnt1-Cre (C57BL/6J)DOI:10.1002/dvdy.20611B6.Cg-E2f1tg(Wnt1-cre)2Sor/J
RRID:IMSR_JAX:022501
Strain; strain background (Mus musculus)Plxnb2-/-DOI:10.1523/JNEUROSCI.4710-06.2007Plxnb2tm1Matl
RRID:MGI:4881705		Gift from Roland Friedel
Strain; strain background (Mus musculus)Plxnb2 cKODOI:10.1523/JNEUROSCI.5381-06.2007
AntibodyAnti-Calbindin D-28k (Rabbit antiserum)SwantCat# CB38,
RRID:AB_2721225		IF(1:1000),
AntibodyAnti-Calbindin D-28k (Mouse monoclonal)SwantCat# 300,
RRID:AB_10000347		IF(1:1000)
AntibodyAnti-Foxp2 (N16) (Goat polyclonal)Santa CruzCat# sc-21069,
RRID:AB_2107124		IF(1:1000)
AntibodyAnti-Glial fibrillary acidic protein (GFAP) (Mouse monoclonal)MilliporeCat# MAB360, RRID:AB_11212597IF(1:500)
AntibodyAnti-Green Fluorescent Protein (GFP) (Rabbit polyclonal)ThermoFisher ScientificCat# A-11122, RRID:AB_221569IF(1:2000)
AntibodyAnti-Green Fluorescent Protein (GFP) (Chicken polyclonal)AvesCat# GFP-1010, RRID:AB_2307313IF(1:2000)
AntibodyAnti-Phospho-Histone H3(Ser10) (Rabbit polyclonal)Cell SignalingCat# 9701; RRID:AB_331535IF(1:1000)
AntibodyAnti-Ki67 (Rabbit polyclonal)AbcamCat# Ab15580; RRID:AB_443209IF(1:500)
AntibodyAnti-Olig-2 (Rabbit monoclonal)MilliporeCat# AB9610;
RRID:AB_10141047		IF(1:500)
AntibodyAnti-Pax6 (Rabbit polyclonal)MilliporeCat# AB2237;
RRID:AB_1587367		IF(1:1000)
AntibodyAnti-Plexin-B2 (Armenian Hamster monoclonal)NovusCat# NBP1- 43310;
RRID:AB_10006672		IF(1:1000)
AntibodyAnti-Sema6A (Goat polyclonal)R and D systemsCat# AF1615;
RRID:AB_2185995		IF(1:500)
AntibodyAnti-Contactin-2/TAG1 (Goat polyclonal)R and D systemsCat# AF4439;
RRID:AB_2044647		IF (1:500)
AntibodyAnti- beta-Tubulin III (Rabbit polyclonal)Sigma-AldrichCat# T2200;
RRID:AB_262133		IF (1:1000)
AntibodyAnti- VGLUT1 (Guinea pig polyclonal)MilliporeCat# AB5905;
RRID:AB_2301751		IF (1:3000)
AntibodyDonkey Anti-Rabbit IgG (H + L) Alexa Fluor 488Jackson ImmunoresearchCat# 711-545-152;
RRID:AB_2313584		IF (1:750)
AntibodyDonkey Anti-Rabbit IgG (H + L) Cy3Jackson ImmunoresearchCat# 711-165-152; RRID:AB_2307443IF (1:750)
AntibodyDonkey Anti-Rabbit IgG (H + L) Alexa Fluor 647Jackson ImmunoresearchCat# 711-605-152; RRID:AB_2492288IF (1:750)
AntibodyBovine Anti-Goat IgG (H + L) Alexa Fluor 647Jackson ImmunoresearchCat# 805-605-180; RRID:AB_2340885IF (1:750)
AntibodyDonkey Anti-Goat IgG (H + L) Cy3Jackson ImmunoresearchCat# 705-165-147; RRID:AB_2307351IF (1:750)
AntibodyDonkey Anti-Mouse IgG (H + L) Alexa Fluor 647Jackson ImmunoresearchCat# 715-605-150; RRID:AB_2340862IF (1:750)
AntibodyDonkey Anti-Chicken IgG (H + L) Alexa Fluor 488Jackson ImmunoresearchCat# 703-545-155; RRID:AB_2340375IF (1:750)
AntibodyDonkey Anti-Chicken IgY (H + L) Cy3Jackson ImmunoresearchCat# 703-165-155; RRID:AB_2340363IF (1:750)
AntibodyGoat Anti-Armenian Hamster IgG (H + L) Alexa Fluor 488Jackson ImmunoresearchCat# 127-545-160; RRID:AB_2338997IF (1:750)
AntibodyGoat Anti-Armenian Hamster IgG (H + L) Cy3Jackson ImmunoresearchCat# 127-165-160; RRID:AB_2338989IF (1:750)
AntibodyDonkey Anti-Guinea Pig IgG (H + L) FITCJackson ImmunoresearchCat# 706-095-148; RRID:AB_2340453IF (1:750)
AntibodyDonkey Anti-Guinea Pig IgG (H + L) Cy3Jackson ImmunoresearchCat# 706-165-148; RRID:AB_2340460IF (1:750)
AntibodyDonkey Anti-Goat IgG (H + L) Alexa Fluor 488Thermo Fisher ScientificCat# A-11055; RRID:AB_25341020IF (1:750)
AntibodyDonkey Anti-Goat IgG (H + L) Alexa Fluor 555Thermo Fisher ScientificCat# A21432; RRID:AB_2535853IF (1:750)
AntibodyDonkey Anti-Mouse IgG (H + L) Alexa Fluor 488Thermo Fisher ScientificCat# A21202; RRID:AB_141607IF (1:750)
OtherHoechst 33342Thermo Fisher ScientificCat# H3570IF (1:1000)
Commercial assay or kitClick-iT EdU Cell Proliferation Kit for Imaging,Alexa Fluor 647 dyeThermo Fisher ScientificCat# C10340
Chemical compound, drugGelatinVWR ChemicalsCat# 24350.262 CAS Number: 9000-70-8
Chemical compound, drugThimerosalSigma-AldrichCat# T8784-5g CAS Number: 54-64-8
Chemical compound, drugTritonX-100Sigma-AldrichCat# X100-500ml CAS Number: 9002-93-1
Chemical compound, drugSYBR Gold nucleic acid stainThermo Fisher ScientificThermo Fisher Scientific:S11494
Software, algorithmFijiNIHRRID:SCR_002285Analysis
Software, algorithmGraphPad PrismGraphPadRRID:SCR_002798Analysis
Software, algorithmImarisOxford InstrumentsRRID:SCR_007370Analysis
Software, algorithmiMovieApplehttp://www.apple.com/fr/imovie/Analysis

Additional files

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